Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Abstract

The function of each renal tubule segment depends on the genes expressed therein. High-throughput methods used for global profiling of gene expression in unique cell types have shown low sensitivity and high false positivity, thereby limiting the usefulness of these methods in transcriptomic research. However, deep sequencing of RNA species (RNA-seq) achieves highly sensitive and quantitative transcriptomic profiling by sequencing RNAs in a massive, parallel manner. Here, we used RNA-seq coupled with classic renal tubule microdissection to comprehensively profile gene expression in each of 14 renal tubule segments from the proximal tubule through the inner medullary collecting duct of rat kidneys. Polyadenylated mRNAs were captured by oligo-dT primers and processed into adapter-ligated cDNA libraries that were sequenced using an Illumina platform. Transcriptomes were identified to a median depth of 8261 genes in microdissected renal tubule samples (105 replicates in total) and glomeruli (5 replicates). Manual microdissection allowed a high degree of sample purity, which was evidenced by the observed distributions of well established cell-specific markers. The main product of this work is an extensive database of gene expression along the nephron provided as a publicly accessible webpage (https://helixweb.nih.gov/ESBL/Database/NephronRNAseq/index.html). The data also provide genomewide maps of alternative exon usage and polyadenylation sites in the kidney. We illustrate the use of the data by profiling transcription factor expression along the renal tubule and mapping metabolic pathways.

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

“…activating DHHCs (41). We examined whether an ENaC-activating DHHC (DHHC 3) is expressed in principal cells of the mouse ASDN, where ENaC and aquaporin 2 are localized.…”

“…We also observed a decrease in TER after treatment of mCCD cl1 cells with 2-BP, which may reflect reduced palmitoylation of claudins normally found within the tight junctions (54 -56). Transcripts for 13 claudins were identified in dissected rat kidney CCDs by deep sequencing (41). A role of claudin palmitoylation in modifying TER was noted in studies of polarized Madin-Darby canine kidney cells, where TER was increased 5-fold after transfection with wild type claudin-14.…”

Genetic-Variation-Driven Gene-Expression Changes Highlight Genes with Important Functions for Kidney Disease

“…Using the protein atlas 59 and Illumina Body Map (GEO: GSE30611), we found that the reproductive tract and the kidney both have intermediate expression of MANBA (not shown), and MANBA is expressed in lysosome of kidney tubule cells ( Figure 4A). 60 Segment-specific expression data obtained from rat kidneys 61 indicate that Manba is expressed in the loop of Henle, with the highest expression level in medullary collecting duct segment.…”

Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ

“…All 14-3-3 isoforms, except , were detected in mouse kidney and mpkCCD 14 cells. The absence of corresponds with recent large scale RNA sequencing studies of isolated kidney segments from rat (12) or cDNA arrays of isolated rat IMCD (39). Although redundancy among 14-3-3 isoforms has been debated (40), 14-3-3⑀, -␤, -, and -had distinct distributions in mouse IMCD cells, suggesting that different isoforms fulfill different roles in these cells.…”

“…Refs. [11][12][13], including the principal cells of the kidney collecting duct (14). The primary functions of these cells are to regulate NaCl (via the epithelial sodium channel, ENaC) 2 and water (via the water channel aquaporin-2, AQP2) transport from the preurine back to the blood and help maintain body NaCl and water homeostasis.…”